Mutant RB1 enhances therapeutic efficacy of PARPis in lung adenocarcinoma by triggering the cGAS/STING pathway

Poly (ADP-ribose) polymerase inhibitors (PARPis) are approved for cancer therapy according to their synthetic lethal interactions, and clinical trials have been applied in non–small cell lung cancer. However, the therapeutic efficacy of PARPis in lung adenocarcinoma (LUAD) is still unknown. We explored the effect of a mutated retinoblastoma gene (RB1) on PARPi sensitivity in LUAD. Bioinformatic screening was performed to identify PARPi-sensitive biomarkers. Here, we showed that viability of LUAD cell lines with mutated RB1 was significantly decreased by PARPis (niraparib, rucaparib, and olaparib). RB1 deficiency induced genomic instability, prompted cytosolic double-stranded DNA (dsDNA) formation, activated the cGAS/STING pathway, and upregulated downstream chemokines CCL5 and CXCL10, triggering immune cell infiltration. Xenograft experiments indicated that PARPi treatment reduced tumorigenesis in RB1-KO mice. Additionally, single-cell RNA sequencing analysis showed that malignant cells with downregulated expression of RB1 had more communications with other cell types, exhibiting activation of specific signaling such as GAS, IFN response, and antigen-presenting and cytokine activities. Our findings suggest that RB1 mutation mediates the sensitivity to PARPis through a synthetic lethal effect by triggering the cGAS/STING pathway and upregulation of immune infiltration in LUAD, which may be a potential therapeutic strategy.


Single-cell RNA sequencing data processing and analysis
The scRNA-seq data of nine samples collected from eight lung adenocarcinoma (LUAD) patients were obtained from GSE171145.The BD Rhapsody system was used to capture the transcriptomic information of the sample-derived single cells.For cell-quality filtering, we removed cells with < 200 expressed genes and a mitochondria UMI rate of > 40%, and mitochondrial genes were removed from the expression table.In order to remove batch effects among samples while preserving biological variation, we used R package "harmony" (v 0.1.0)to integrate cells across individuals.First, we searched highly variable genes using a variance-stabilizing transformation method from the "Seurat" (v 4.1.0)package in R. Top 2000 highly variable genes were selected for principal component analysis (PCA).Then, we ran "Harmony" on the first 30 PCs, and performed clustering by the smart local moving (SLM) algorithm with a resolution of 0.4, obtained the corrected PC embeddings finally.Cells were visualized using a 2-dimensional tSNE on the same distance metric.
Cell type initial annotation was performed with "SingleR" (v 1.8.1) package using HumanPrimaryCellAtlasData provide by "celldex" (v 1.4.0)package, then the cell type annotation was adjusted by the expression of canonical cell type marker genes.
Another processed scRNA-seq data with 11 early stage (stage I, stage II, and stage III) LUAD patients (tLung) and four advanced stage (stage IV) LUAD patients (tL/B) were obtained from GSE131907.Cells were subjected to scRNA-seq using 10x Genomics.The raw gene-cell-barcode matrix was performed quality control and lognormalized to TPM-like values (log2 [TPM + 1]).Then, scRNA-seq data were clustered, followed by annotating according to known marker genes using Seurat algorithm.
To detect malignant cancer cells, we performed CopyKAT algorithm by R package "copykat" (v 1.0.8) to estimate genomic copy number profiles using gene expression profiles.Epithelial cells which labeled as "aneuploid" were considered as malignant cancer cells, and further reserved malignant epithelial cells in which retinoblastoma tumor suppressor gene (RB1) expressed (expression value non-zero).

The cell-cell communication among ME-RB1-H, ME-RB1-L and other type cells
were analyzed using the R package "CellChat" (v1.4.0).We focused on the human database in CellChat and identified over-expressed ligands or receptors by "identifyOverExpressedGenes" and "identifyOverExpressedInteractions" functions.
Then, we mapped gene expression data onto PPI network by "projectData" function.
"computeCommunProb" and "filterCommunication" functions (min.cells= 10) were used to compute communication probability and infer cellular communication network."computeCommunProbPathway" and "aggregateNet" functions were used to infer the cell-cell communication at a signaling pathway level between each cell type.
We identified the communications between ME-RB1-L cells and other cell types (excluded ME-RB1-H cells), and the communications did not appear in ME-RB1-H cells simultaneously, defined as specific communications in ME-RB1-L cells.

Survival analysis
Overall survival data were downloaded from TCGA.LUAD patients with RB1 mutation were grouped according to the median expression of PARP1.Log-rank test was used to assess the difference in the survival time between two group patients.
Kaplan-Meier plots were used to present the results.

CRISPR/Cas9-mediated RB1 knockout cell line
For RB1 knockout (RB1-KO), the single guide RNAs (sgRNAs) were designed using the online CRISPR design tool (Red Cotton TM , Guangzhou, China, https://en.rc-crispr.com/).The exon 1 region of RB1 was selected to be targeted by CRISPR/Cas9 genome editing.A ranked list of sgRNAs was generated with specificity and efficiency scores.The pair of oligos for two targeting sites were annealed and ligated to the YKO-RP006 vector (Ubigene Biosciences Co., Ltd.,

TUNEL staining
TUNEL assay was performed to detect apoptotic cells using an in situ cell death detection kit (fluorescein, Roche Applied Science) according to the manufacturer's instructions.Cell slides were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) and permeabilized with 0.1% Triton X-100 in PBS.The treatment group was mixed with 50u1 TdT and 450ul labeled dUTP solution, and the reaction was performed for 60 min at 37°C in a humidified atmosphere in the dark.DAPI was prepared to stain nuclei.The number of TUNEL-positive cells (green cells) and the total number of cells (blue cells) were measured by Image-Pro Plus.

Western blot
Cells were lysed in RIPA buffer with a cocktail of phosphatase inhibitor and protease inhibitor.

Quantitative real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.The concentration and purity of RNA were detected by NanoDrop 8000 (Thermo, USA) and reverse transcribed to cDNA using All-in-One First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China, AT341-02).qRT-PCR was performed by the ABI QS6 system (Applied Biosystems, ABI, USA).The data was analyzed by 2 −ΔΔCt and normalized to Actb in each sample.
The following primer sequences were used:

Immunofluorescence staining
Cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature before staining and permeabilized with 0.4% Triton X-100 in PBS for 30 minutes.
After 3 additional washes in phosphate-buffered saline (PBS) for 5 min, cells were blocked with Goat Serum (Boster, California, USA) for one hour at room temperature.

PicoGreen staining
PicoGreen staining was performed using Quant-iT Pico-Green dsDNA reagent kits from Thermo Fisher Scientific.Cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature and permeabilized with 0.4% Triton X-100 in PBS for 30 minutes.PicoGreen was diluted with a cell culture medium at a dilution ratio of 1: 500 at 37°C for one hour.Fluorescence images were collected using the Zeiss Confocal Laser Scanning Microscope 800 (Carl Zeiss, Oberkochen, Germany).DAPI was used to mark nuclei.